IJBPAS, November, 2015, 4(11), Special Issue: 01-11 ISSN: 2277–4998

INVESTIGATING THE KARYOTYPE OF FIVE POPULATIONS OF “PLANTAGO MAJOR”SPECIES USING THE IMAGE ANALYSIS SYSTEM

FATEMEH AGHAJANI1, ARMAN MAHMOUDI OTAGHVARI2*, SADEGH POURMORADI3, SEDIGHEH KELIJ4 1: M.Sc. Student, Department of Biology, University of Mazandaran, , 2: Department of Biology, University of Mazandaran, Babolsar, Iran 3: Mazandaran Agricultural Research and Natural Resources Center, Mazandaran, Iran 4: Department of Biology, University of Mazandaran, Babolsar, Iran *Corresponding Author: E Mail: [email protected] ABSTRACT With the aim of investigating the Cytotaxonomy of a species of Plantago genus from the family of Plantaginaceae, collected from the habitats of the northern , the karyotype of 5 populations belonging to P.major species is studied in the meristem cells of root tip using an image analysis system. The chromosomal dimensions such as the length of short arm and the length of the whole chromosome are measured and the AR and CI statistics are also calculated after preparation, staining, and microscopic investigation of samples and separation of chromosomes by Photoshop software through Micromeasure software in at least 3 appropriate cells of mitotic metaphase. The populations are measured based on the karyotype statistics including S%, %TF, A1, DRL, A2, VRC, Levan karyotype formula and grouping the Stebbins for studied populations. In this research, the studied populations of P.major had the chromosomal number of 2n=2x= 12 and the basic chromosome number was equal to x=6 in all populations and it was consistent with previous studies and this indicates the diploid species in Iran. Furthermore, according to the Levan Table, the chromosomes of studied populations were from the metacentric and submetacentric species. Keywords: Plantago major , Cytogenetic Diversity, Chromosome, Karyotype, Northern Iran INTRODUCTION Plantago genus has about 483 species [21], perennial species for it. It has the dense and 22 species of this genus have been reported alternate Rosulates. The male and female from Iran in Flora Iranica (Mozaffarian, flowers are inside the spikes until a narrow 1994). Most of its plants are have one or and extremely long cylinder with a few or a multiple years of age and there are a few lot of flowers [11],The application of

1 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article cytogenetic information in systematic and population of a species because different phylogenetic nature of this genus has been populations of a species show their unique considered from previous years. Rana genomic consistency with the environment (1996) has offered a new suggestion of wherein they grow, so that it is believed classifying the Plantago genus based on the that the chromosomal analyses along with cytology knowledge and classified this the genetic and morphological studies can genus into 6 subgenus including Coronopus be the reliable indices in assessing the Rahn, Albicans Rahn, Plantago major, relative relationship between the species of Psyllium Juss, Littorella Rahn, and a genus [8],The investigation of difference Bougueria Rahn, and has indicated that in the number of chromosome, its Plantago genus has 3 different chromosome morphology as well as the difference of bases including x= 4, x= 5, and x = 6. ploidy level are utilized as the evidence for The cultivation of this genus has been more convergence between the species and taken into account due to the important somatic hybrids derived from the cell components (they are more applied in fusion [13],A lot of international studies are pharmacy) in order to extract the active conducted by different researchers on compounds [7],Plantago major has the determining the chromosome number and biological active compounds like ploidy level of species and subspecies of a Polysaccharide, Glycoside, Lipid and Plantago genus. McCullagh [14],has Terpenoid, and Sorbitol [18], It has the presented the first chromosome counting astringent, anti-diarrhea, sweat-inducing, and morphology of chromosomes. Gorenf, diuretic, and anti-rheumatism effects Vasudevan and Favarger (1972) studied the (Zargari, 1992). It is effective in curing the polyploidy in this species. Santosh and colic, jaundice, and liver diseases (Beara, Raghbir (2011) investigated the 2000). Furthermore, it has the anti- chromosome number of some species of inflammatory on the liver. (Turel et al, Plantago genus in the northern India. 2009) and it is especially applied in wound Conducting the Cytotaxonomic study of healing[2], Plantago major is spread in this genus in the northern Iran, we will be different parts of the Earth and grows in a able to provide the more accurate and large area of Europe, Asia, and North detailed classification of this Taxon and America [22],and [6],It is growing in provide the better and more effective key of almost all regions of Iran. It is essential to identification by discovering the conduct the karyotype studies in the chromosomal status of plantago major

2 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article species populations in the north of country 16-18 hours for stabilization of internal in addition to determining the level of structure of cell and maintaining the karyotype symmetry. original status as well as HCl 1N for MATERIALS AND METHODS hydrolysis for 5-10 minutes at 60°C and This study is conducted on five accessions also ethyl alcohol 70% for storing the roots from P. major species in different regions for a long time in addition to hematoxylin of northern Iran (Gorgan, Behshahr, Sari, staining solution in order to investigate the Babolsar, and ). The studied seeds shape and structure of chromosomes. germinate after disinfection by bleach 15% Afterwards, the apical meristem of roots is for ten minutes and putting them on the wet separated and the chromosomal images are filter paper inside the Petri dishes at 25 °C, taken by adding acetic acid 45% and and the root tips are separated from the through the squash method (Agayev, 1996). seeds when the root lengths reach 0.5-1 cm The chromosomes of each cell are and are put as the pretreatment in solution separated by Photoshop software and of Alpha Bromonaphthalene 1% for 2 arranged on a page, and then the length of hours. The pretreatment refers to stopping long arm, length of short arm, and total the cell division at the metaphase stage. length of chromosome are measured in at The next stages of this study apply the least 3 appropriate mitotic metaphase cells compounds such as the Farmer solution for through Micromeasure software.

Table 1: Characteristics of species and populations applied in this research Species Collection place Height above sea level P.major Mazandaran - Sari 1900 P.major Mazandaran - Babol 2577 P.major Mazandaran - Behshahr 400-660 P.major Gorgan 3500 P.major Mazandaran - Babolsar -21 The data is analyzed in the form of a characteristics including the length of long completely randomized unbalanced and short arms are calculated for different method, and then the mean of each populations through Excel software and the characteristic is compared by Duncan test ideogram of each population drawn. The at the level of 5%. The karyotype statistics chromosomes are arranged based on the S%, %TF (Huziwara, 1962), A1, DRL, A2 reduced length of chromosome and the (Romrozarco, 1971), Levan karyotype centromeres aligned in drawing the formula (1962) and Symmetry Class ideogram. (Stebbins, 1971) are measured for studied RESULTS populations and the populations compared The mitotic metaphase images of studied with each other. The mean chromosomal species and populations are presented in

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Figure 1. All of the chromosomes are not and short arms are calculated through Excel clearly seen in an image since they are software and the ideogram of each one multilevel. The mean chromosomal drawn. (Figure 1) characteristics including the length of long

Babol P.major

Sari P.major

Gorgan P.major

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Behshahr P.major

Babolsar P.major

Figure 1: The mitosis metaphase image and karyogram of studied populations of Plantago major species In drawing the ideogram of species, the difference between the differences in the chromosomes are arranged based on relative length (DRL) of chromosomes, reducing the total chromosome length and Pearson dispersion coefficient (A2) as the the centromeres placed in the same inter-chromosomal asymmetry index, direction. The parameters including the Value of Relative Chromatin (VRC), Levan relative length of shortest chromosome karyotype formula and Stebbins grouping (S%), the total form percentage (%TF), A1 are measured for species and presented in (intra-chromosomal asymmetry index), the Table 2.

Table 2: Measurement parameters of karyotype symmetry for five studied populations from Plantago major species Karyotype Population %S %TF A1 DRL A2 VCR SC formula Babol 14.21 38.40 0.36 4.78 0.10 4.05 1A 5m + 1sm Sari 13.54 39.51 0.348 6.87 0.14 3.51 1A 6m Behshahr 14.46 38.87 0.35 4.62 0.21 3.78 1A 5m + 1sm Babolsar 12.63 38.31 0.38 7.56 0.20 3.32 1A 6m Gorgan 13.27 36.46 0.42 5.94 0.13 4.25 1A 5m + 1sm

The results indicate that Babol proportion chromosomal base is x=6. The lengths of has 2n= x2x=12 chromosomes and its its longest and shortest chromosome are

5 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article equal to 4.61 and 3.46 respectively. In this arms 14.97 μm, and the total length of short species, the maximum and minimum arms 9.33 μm. This species with the lengths of long arm (L) are equal to 2.04 karyotype formula of 5m+1sm is in class and 2.88, the lengths of short arm (S) equal A1 in terms of karyotype symmetry and to 1.40 and 1.73, the ratio of lengths of long based on Stebbins table, and the average to short arm of chromosome (AR) equal to length of each chromosome is 4.05 μm. Its 2.15 and 1.26, the centromeric indexes (CI) A1 is equal to 0.36, A2 0.12, S% equal to 0.31 and 0.44, and the percentages of 14.24, %TF equal to 38.99 and DRL equal relative chromosome length equal to 18.99 to 4.78 (Table 2). The ideogram of and 14.31. Furthermore, in this species, the population for this species is presented in length of a set of chromosomes (haploid) is Figure 2. equal to 24.31 μm, the total length of long

Figure 2: Ideogram of Babol population from Plantago major species The study on Sari population indicates that chromosomes (haploid) is equal to 21.09 the lengths of its longest and shortest μm, the total length of long arms 12.75 μm chromosomes are equal to 4.30 and 2.85 and the total length of short arms 8.33 μm. respectively. In this species, the maximum This species with the karyotype formula of and minimum lengths of long arm (L) are 6m is in A1 Class based on Stebbins table 2.43 and 1.68, the lengths of short arm (S) in terms of karyotype symmetry, and the 1.87 and 0.161, the ratios of lengths of long average length of each chromosome is to short arms of chromosome (AR) equal to equal to 3.54 μm. Its 1A is equal to 0.34, 1.30 and 1.72, the centromeric indexes (CI) A2 equal to 0.14, S% 13.54, %TF 39.54 0.36 and 0.43, and the relative lengths of and DRL 4.62 and these are presented in chromosome are equal to 20.4 and 13.56. In Table 2. The ideogram of population for this species, the length of a set of this species is presented in Figure 3.

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Figure 3: Ideogram of Sari population from Plantago major species The study on Babolsar population indicates chromosomes (haploid) is equal to 19.92 that the lengths of its longest and shortest μm, the total length of long arms 12.29 μm chromosomes are equal to 4.02 and 2.52 and the total length of short arms 7.63 μm. respectively. In this species, the maximum This species with the karyotype formula of and minimum lengths of long arm (L) are 3m+3sm is in A1 Class based on Stebbins 238 and 1.60, the lengths of short arm (S) table in terms of karyotype symmetry, and 1.65 and 0.92, the ratios of lengths of long the average length of each chromosome is to short arms of chromosome (AR) equal to equal to 3.32 μm. Its 1A is equal to 0.38, 1.44 and 1.74, the centromeric indexes (CI) A2 equal to 0.16, S% 12.64, %TF 38.31 0.36 and 0.41, and the relative lengths of and DRL 7.56 and these are presented in chromosome are equal to 20.20 and 12.64. Table 2. The ideogram of population for In this species, the length of a set of this species is presented in Figure 4.

Figure 4: Ideogram of Babolsar population from Plantago major species Plantago major species in Behshahr respectively. In this species, the maximum proportion has 2n= x2x=12 chromosomes and minimum lengths of long arm (L) are and its chromosomal base is x=6. The equal to 2.24 and 2.15, the lengths of short lengths of its longest and shortest arm (S) equal to 1.01 and 1.88, the ratio of chromosome are equal to 4.32 and 3.28 lengths of long to short arm of chromosome

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(AR) equal to 2.25 and 1.30, the karyotype formula of 5m+1sm is in class centromeric indexes (CI) 0.30 and 0.43, A1 in terms of karyotype symmetry and and the percentages of relative based on Stebbins table, and the average chromosome length equal to 19.22 and length of each chromosome is 3.78 μm. Its 14.60. Furthermore, in this species, the A1 is equal to 0.35, A2 0.21, S% equal to length of a set of chromosomes (haploid) is 14.46, %TF equal to 38.87 and DRL equal equal to 22.72 μm, the total length of long to 4.62 (Table 2). The ideogram of arms 13.67 μm, and the total length of short population for this species is presented in arms 8.83 μm. This species with the Figure 5.

Figure 5: Ideogram of Behshahr population from Plantago major species The studies on Gorgan population indicates 13.27. Furthermore, in this species, the that the Plantago major species in has 2n= length of a set of chromosomes (haploid) is x2x=12 chromosomes and its chromosomal equal to 25.55 μm, the total length of long base is x=6. The lengths of its longest and arms 16.23 μm, and the total length of short shortest chromosome are equal to 4.90 and arms 9.31 μm. This species with the 3.39 respectively. In this species, the karyotype formula of 5m+1sm is in class maximum and minimum lengths of long A1 in terms of karyotype symmetry and arm (L) are equal to 3.34 and 2.23, the based on Stebbins table, and the average lengths of short arm (S) equal to 1.15 and length of each chromosome is 4.25 μm. Its 1.88, the ratio of lengths of long to short A1 is equal to 0.42, A2 0.13, S% equal to arm of chromosome (AR) equal to 2.13 and 13.27, %TF equal to 36.46 and DRL equal 1.43, the centromeric indexes (CI) 0.31 and to 5.94 and these are presented in Table 2. 0.41, and the percentages of relative The ideogram of population for this species chromosome length equal to 19.21 and is presented in Figure 6.

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Figure 6: Ideogram of Gorgan population from Plantago major species CONCLUSION chromosomal number of this species equal to In this study, Plantago major species is diploid 2n=2x=8. Mohsenzadeh et al (2008) reported and has (2n=12) chromosomes. The same the chromosomal number of Plantago major research is conducted by McCullagh [14],and equal to 2x=12 in Iran. Bocher (1955) and Rahan [16],on P. major The species and population with the shortest species and the chromosomal number and relative length of chromosome (S%) has more ploidy level of this species equal to (2n= 2x= symmetric karyotype. In this study and among 12). Furthermore, Favarger and Vasudevan the population of studied species, Babolsar (1972) reported the chromosomal number of population with the value of 12.63 has the this species equal to 2n=6x= 36 in their studied maximum value of this statistic and the most in Himalayas. Moreover, Briggs [3],and Jain symmetric karyotype (Table 2). The more %TF (1978) reported that this species is Hexaploid is close to 50, the more it has the symmetrical according to the chromosome counting in Italy. karyotype. According to Table 2, the highest Santosh and Raghir (2011) reported the percentage equal to 39.54 belongs to Sari chromosomal number of P. major species equal population. In other words and according to this to (2n=12.24) according to the cytological statistics, P.major of Sari population has the analysis of pollen meiosis in the northern India. most symmetric karyotype among the According to the polymorphic study by populations. Gorgan population with the value Favarger and Vasudeva (1972) on this species, of 36.46 has the least amount of this statistics the studies indicate that the changes of and the most asymmetric karyotype. The value quantitative survey reflect a change in the of A1 as the index of intra-chromosomal number of genes (phylogenetic). Badra and El- asymmetry is lower among the metacentric kholy [1],reported the chromosomal number chromosomes, so that if all chromosomes of a and karyotype of Plantago genus equal to species are Meta (M), the value of A1 will be 2n=12 in Egypt. Ebadi et al [5],investigated the equal to zero, and thus it has the highest degree karyotype and karyomorphological changes in of karyotype symmetry. Based on this statistics, Plantago ovata Forsk in Iran and reported the Gorgan population has the highest value and

9 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article most asymmetric karyotype. As shown in Table of Babolsar and Sari population have been from 2, two indexes, A1 and %TF, which show the metacentric (m) type, and Babol, Behshahr, and intra-chromosomal symmetry or asymmetry, Gorgan have metacentric and submetacentric have inverse relationship with each other, so types. According to Stebbins table, all that the more %TF is increased, the more the populations of studied species have been in value of As is reduced. In terms of DRL, since class A1. In general, according to karyotype the lower values of this statistic indicate the statistics, namely, %S, %TF, A1, DRL and A2, higher symmetry of karyotype among the Behshahr, Gorgan and Babol populations have studied species, Sari population with the value the most asymmetric karyotype among the of 4.62 has more symmetric karyotype than populations of species and the higher degree of other populations. Babolsar population with karyotype evolution. Accordingly, Sari and DRL of 7.56 has the most asymmetric Babol populations have the most symmetric karyotype among the studied species (Table 2). karyotype among the studied populations The higher value of this statistic in a species or (Table 2). cultivar indicates that the karyotype is more REFERENCES asymmetric and that species or cultivar is in a 1. Badr, A., & El-Kholy, M. A. (1987). higher degree of karyotype evolution. Pearson Chromosomal studies in the Egyptian dispersion coefficient (A2), as the index of Flora II. Karyotype studies in the genus intra-chromosomal asymmetry, shows the Plantago L. Cytologia, 52(4), 725-731. karyotype asymmetry in terms of relationship 2. Brara, I.N., D.Z. ORCIC, MM. Lesjak, between the sizes of different chromosomes. N.M., Mimica-Dukic, B.A. Pekovik The more the value of A2, the more the and M.R. Popovic.2010 Liquid difference between the length of chromosomes, Chromatography/tandem mass the more asymmetric karyotype and thus the spectrometry study of anty- species more evolved. Accordingly, Behshahr inflammatory activity of Plantain species with statistic equal to 0.21 has the most (Plantago L.). asymmetric karyotype among the population of 3. Briggs, G. B. 1973. Chromosomal studied species and Babol population has the studies on Plantago in Australia. lowest value of this statistic equal to 0.10 and Contrib. New S. Wales, Nation. Herb. the most symmetric karyotype. Among the 4: 399-405. studied populations, Gorgan and Babolsar with 4. Cartier, D. 1971. Etude the statistics of 4.25 and 3.32 microns have the biosystematique de quelques especes highest and lowest Value of Relative du genre Plantago (Tourn.) L. (Sections Chromatin (VRC) respectively. On the basis of Coro nopus D.C. et Oreades Decne.) I. karyotype formula and based on the table Historiques races 5.Chromosomiques presented by Levan, the studied chromosomes de Plantago alpina L. et de Plan tago

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